1. Field of the Invention
The present invention relates to the identification of compounds in mass spectrometry; and more specifically, the invention addresses the assignment of weighting factors to ion adducts that populate collected spectra.
2. Description of Related Art
A mass spectrometer (MS) is one of the most powerful tools available for quantitative and qualitative analysis of an unknown or suspect compound. One type of mass spectrometer, available since the 1950's, employs electron impact (EI) ionization and fragmentation—whereby vaporized molecules are simultaneously ionized and fragmented with high energy under high vacuum to produce a broad spectrum of ions. EI-MS is typically coupled to a gas chromatograph (GC) to insure adequate molecule separation prior to analysis. Spectra obtained by this technique provide unique spectral “fingerprints” of molecules that are reproducible under set conditions, e.g., 70 eV electron energy and approximately unit resolution.
With the increasing power and availability of computers, large collections, or libraries, of EI-MS spectra have been developed along with associated searching software. Such databases, which may contain hundreds of thousands of molecular fingerprints, are often purchased with the instrument. A typical library search compares the spectrum of an unknown compound with the spectra of known compounds in the library and retrieves “hits” of compounds that have similar spectra.
Unfortunately, GC separation, which is the separation technique usually employed with EI-MS, is not possible with a significant percentage of molecules. GC separation requires that molecules be injected as a vapor. Thus, large, thermally labile molecules are not always suitable for GC analysis. In addition, GC analysis, as often as not, requires extensive and time consuming sample preparation and derivatization. This is especially true in the case of compounds with high polarity and low volatility.
In contrast, liquid chromatography (LC) is a more versatile separation technique. For LC separation, the molecules are injected in solution. Most molecules of interest are more easily solubilized than vaporized. However, LC is not compatible with EI, where the molecule must be introduced into high vacuum as a vapor. Therefore, LC is coupled to mass spectrometers that utilize different mechanisms for ionization, e.g., atmospheric pressure ionization (API).
LC-API-MS is well suited to the investigation of semi-volatile, thermo-labile and polar substances, like pesticides, explosives and forensically relevant substances. Unfortunately, existing EI-MS libraries are not suitable for identifying API-MS spectra. New API-MS libraries must be constructed.
One difficulty in constructing libraries for API-MS is the fact that API, unlike EI, generates little or no fragmentation. It is not uncommon to see only the pseudo molecular ion in the mass spectra. This means that API-MS is an excellent means for identifying the molecular weight of an unknown compound, but it cannot distinguish between the thousands of molecules that have any given molecular weight.
To increase ion fragmentation, collision induced dissociation (CID) is typically used in combination with API-MS. However, CID is highly variable. The ions generated by CID, as well as the ion ratios, can vary between different mass spectrometer models, between instruments of the same model, and between day to day operations on the same instrument. These variations occur, among other things, as a function of the ion source and the highly sensitive nature of the CID region.
One means for addressing the variable nature of API-CID-MS spectra is the use of performance based tuning. Performance based tuning attempts to normalize the difference in CID fragmentation between instruments. However, even when API-CID-MS is used in conjunction with performance based tuning, the fragmentation of the molecules still varies considerably under any given set of conditions. Accordingly, it is known to generate multiple spectra under multiple conditions, which may then be evaluated separately or as part of a composite spectrum that presents the sum or average of the various spectra.
An API MS generated spectrum, or composite spectrum, usually contains a peak for the “pseudo molecular ion,” from which the molecular weight can be correctly inferred. Usually this is a protonated molecular ion [(M+H)]+. However, heavier positively charged adducts are often seen that can confuse the untrained observer. For example, salts, such as ammonium ion adducts [(M+NH4)]+, sodium ion adducts [M+Na]+ and potassium ion adducts (M+K)+ may appear in, or even dominate, the spectrum. In addition, positively charged dimers and oligomers of the parent molecule may appear, e.g., (2M+H)+. These adducts may or may not be present in the spectrum depending upon chromatographic conditions, purity of solvents, pH, etc. The trained analyst can use adducts to confirm the identity of the pseudo molecular ion. But, because adducts are not always present and, when present, vary greatly in abundance, their absence has no particular significance. There remains a need in the art to create methods to identify compounds by API-MS spectra in light of the adducts that may or may not be detected.